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WO2021236869A2 - Procédés de détection de virus - Google Patents

Procédés de détection de virus Download PDF

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Publication number
WO2021236869A2
WO2021236869A2 PCT/US2021/033303 US2021033303W WO2021236869A2 WO 2021236869 A2 WO2021236869 A2 WO 2021236869A2 US 2021033303 W US2021033303 W US 2021033303W WO 2021236869 A2 WO2021236869 A2 WO 2021236869A2
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WIPO (PCT)
Prior art keywords
coronavirus
cov
antigen
sars
antibodies
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PCT/US2021/033303
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WO2021236869A3 (fr
Inventor
Christopher D. HEANEY
Nora PISANIC
Pranay RANDAD
Steve Granger
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SALIMETRICS LLC
Johns Hopkins University
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SALIMETRICS LLC
Johns Hopkins University
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Publication of WO2021236869A3 publication Critical patent/WO2021236869A3/fr
Anticipated expiration legal-status Critical
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/10—Detection of antigens from microorganism in sample from host
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/26—Infectious diseases, e.g. generalised sepsis

Definitions

  • the present invention relates to compositions and methods for accurate determination of coronavirus infection.
  • the SARS-CoV-2 pandemic has caused >4.2 million COVID-19 cases and >289 thousand deaths, as of May 12, 2020, involving all populated continents (WHO. Coronavirus disease 2019 (COVID-19) Situation Report - 70).
  • the SARS-CoV-2 outbreak has expanded from urban to rural areas of the United States with more than 1.3 million people being diagnosed with COVID-19 and more than 81,000 deaths (WHO. Coronavirus disease 2019 (COVID-19) Situation Report - 70).
  • Development of improved antibody assays to detect historical infection with SARS-CoV-2 is an unmet need in the ongoing pandemic response.
  • Disclosed herein are methods for detecting one or more anti-coronavirus antibodies in an oral fluid sample comprising: a) obtaining or having obtained an oral fluid sample from a subject, wherein the oral fluid sample comprises one or more anti-coronavirus antibodies; b) contacting the oral fluid sample with one or more coronavirus antigens under conditions where the one or more coronavirus antigens bind to the one or more anti- coronavirus antibodies to form a detectable complex, wherein each of the one or more antigens are covalently coupled to a bead comprising a detectable label; and c) detecting the presence of the detectable complexes; thereby detecting the presence of the one or more anti- coronavirus antibodies in the oral fluid sample.
  • Disclosed herein are methods for detecting one or more anti-respiratory virus antibodies in a saliva sample comprising: a) obtaining or having obtained an oral fluid sample from a subject, wherein the oral fluid sample comprises one or more anti- respiratory virus antibodies; b) contacting the oral fluid sample with one or more respiratory virus antigens under conditions where the one or more respiratory virus antigens bind to the one or more anti-respiratory virus antibodies to form a detectable complex, wherein each of the one or more antigens are covalently coupled to a bead comprising a detectable label; and c) detecting the presence of the detectable complexes; thereby detecting the presence of one or more anti-respiratory virus antibodies in the oral fluid sample.
  • Disclosed herein are methods for detecting a respiratory virus in an oral fluid sample comprising: a) obtaining or having obtained the oral fluid sample from a subject, wherein the oral fluid sample comprises one or more respiratory viruses; b) contacting the oral fluid sample with one or more antibodies under conditions where the one or more antibodies bind to the one or more respiratory viruses to form a detectable complex, wherein each of the one or more antibodies are covalently coupled to a bead comprising a detectable label; and c) detecting the presence of the detectable complexes; thereby detecting the presence of the respiratory virus in the oral fluid sample.
  • Sino Biol. Sino Biological
  • NAC Native Antigen Company
  • N nucleocapsid protein
  • ECD SI: SI subunit of spike protein
  • S2 S2 subunit of spike protein
  • RBD receptor binding domain
  • MFI mean fluorescence intensity.
  • Sino Biol. Sino Biological
  • NAC Native Antigen Company
  • N nucleocapsid protein
  • ECD SI: SI subunit of spike protein
  • S2 S2 subunit of spike protein
  • RBD receptor binding domain
  • (i): produced in insect cell; Se: Sensitivity; Sp: specificity; MFI mean fluorescence intensity.
  • Sino Biol. Sino Biological
  • NAC Native Antigen Company
  • N nucleocapsid protein
  • ECD SI: SI subunit of spike protein
  • S2 S2 subunit of spike protein
  • RBD receptor binding domain
  • (i): produced in insect cell; Se: Sensitivity; Sp: specificity; MFI mean fluorescence intensity.
  • Fig. 4 shows the heat map detailing the sensitivity and specificity of each saliva SARS-CoV-2 antigen-specific IgG, IgA, and IgM.
  • Samples collected from subjects with RT- qPCR confirmed prior SARS-CoV-2 infection are stratified into samples collected ⁇ 10 days post symptom onset and samples collected >10 days post symptom onset. The average MFI of negative samples + 3 standard deviations was used to set the MFI cut off for each SARS- CoV-2 antigen-specific IgG, IgA, and IgM. Note.
  • Samples collected from subjects with RT- qPCR confirmed prior SARS-CoV-2 infection are stratified into samples collected ⁇ 10 days post symptom onset and samples collected >10 days post symptom onset.
  • the average MFI of negative samples + 3 standard deviations was used to set the MFI cut off for each SARS- CoV-2 antigen-specific IgG, IgA, and IgM. Note.
  • Fig. 6 shows the comparison of saliva and serum SARS-CoV-2 antigen-specific IgG, IgA, and IgM responses vs. days post symptom onset.
  • the trajectory of antibody responses are estimated using a LOESS curve.
  • Sino Biol. Sino Biological
  • NAC Native Antigen Company
  • N nucleocapsid protein
  • ECD SI: SI subunit of spike protein
  • S2 S2 subunit of spike protein
  • RBD receptor binding domain
  • MFI mean fluorescence intensity.
  • Fig. 8 shows that saliva collected at >10 days post symptom onset displayed significantly elevated median MFI compared to negatives for the SARS-CoV-2 antigen- specific IgG tested.
  • Fig. 9 shows that serum collected at >10 days post symptom onset displayed significantly elevated median MFI compared to negatives for the SARS-CoV-2 antigen- specific IgG, IgA, and IgM tested.
  • Ranges can be expressed herein as from “about” or “approximately” one particular value, and/or to “about” or “approximately” another particular value. When such a range is expressed, a further aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about,” or “approximately,” it will be understood that the particular value forms a further aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint and independently of the other endpoint. It is also understood that there are a number of values disclosed herein and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that each unit between two particular units is also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.
  • the terms “optional” or “optionally” mean that the subsequently described event or circumstance may or may not occur and that the description includes instances where said event or circumstance occurs and instances where it does not.
  • sample is meant a tissue or organ from a subject; a cell (either within a subject, taken directly from a subject, or a cell maintained in culture or from a cultured cell line); a cell lysate (or lysate fraction) or cell extract; or a solution containing one or more molecules derived from a cell or cellular material (e.g. a polypeptide or nucleic acid), which is assayed as described herein.
  • a sample may also be any body fluid or excretion (for example, but not limited to, blood, urine, stool, saliva, tears, bile) that contains cells or cell components.
  • the sample can be a saliva sample.
  • the saliva sample can be a clarified saliva sample.
  • the term “subject” can refer to the target of administration, e.g., a human.
  • the subject of the disclosed methods can be a vertebrate, such as a mammal, a fish, a bird, a reptile, or an amphibian.
  • the term “subject” also includes domesticated animals (e.g., cats, dogs, etc.), livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), and laboratory animals (e.g., mouse, rabbit, rat, guinea pig, fruit fly, etc.).
  • a subject is a mammal.
  • a subject is a human.
  • the term does not denote a particular age or sex. Thus, adult, child, adolescent and newborn subjects, as well as fetuses, whether male or female, are intended to be covered.
  • the term “patient” refers to a subject afflicted with a disease or disorder.
  • the term “patient” includes human and veterinary subjects.
  • the “patient” has been identified with a need for testing for suspected coronavirus exposure, such as, for example, prior to obtaining a sample.
  • the term “comprising” can include the aspects “consisting of’ and “consisting essentially of.”
  • SARS virus protein refers to any protein of any SARS virus strain or its functional equivalent as defined herein.
  • the invention includes, but is not limited to, SARS polymerase, the S (spike) protein, the N (nucleocapsid) protein, the M (membrane) protein, the small envelope E protein and their functional equivalents.
  • Epitope refers to an antigenic determinant of a polypeptide.
  • An epitope could comprise three amino acids in a spatial conformation which is unique to the epitope. Generally, an epitope consists of at least five such amino acids, and more usually consists of at least 8-10 such amino acids. Methods of determining the spatial conformation of such amino acids are known in the art.
  • polypeptide refers to any peptide, oligopeptide, polypeptide, gene product, expression product, or protein. A polypeptide is comprised of consecutive amino acids.
  • polypeptide encompasses naturally occurring or synthetic molecules.
  • amino acid sequence refers to a list of abbreviations, letters, characters or words representing amino acid residues. Amino acids may be referred to herein by either their commonly known three letter symbols or by the one- letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.
  • isolated polypeptide or “purified polypeptide” is meant a polypeptide (or a fragment thereol) that is substantially free from the materials with which the polypeptide is normally associated in nature.
  • the polypeptides of the invention, or fragments thereof can be obtained, for example, by extraction from a natural source (for example, a mammalian cell), by expression of a recombinant nucleic acid encoding the polypeptide (for example, in a cell or in a cell-free translation system), or by chemically synthesizing the polypeptide.
  • polypeptide fragments may be obtained by any of these methods, or by cleaving full length polypeptides.
  • an antibody recognizes and physically interacts with its cognate antigen and does not significantly recognize and interact with other antigens; such an antibody may be a polyclonal antibody or a monoclonal antibody, which are generated by techniques that are well known in the art.
  • the term “neutralize” refers to the ability of an antibody, or antigen binding fragment thereof, to bind to an infectious agent, such as coronavirus, and reduce the biological activity, for example, virulence, of the infectious agent.
  • An antibody can neutralize the activity of an infectious agent, at various points during the lifecycle of the virus.
  • an antibody may interfere with viral attachment to a target cell by interfering with the interaction of the virus and one or more cell surface receptors.
  • an antibody may interfere with one or more post-attachment interactions of the virus with its receptors, for example, by interfering with viral internalization by receptor-mediated endocytosis.
  • an antigen can refer to a compound, composition, or substance that can stimulate the production of antibodies or a T-cell response in an animal, including compositions that are injected or absorbed into an animal.
  • An antigen reacts with the products of specific humoral or cellular immunity, including those induced by heterologous immunogens.
  • an antigen can be a virus antigen, such as a coronavirus antigen.
  • determining can refer to measuring or ascertaining an activity or an event or a quantity or an amount or a change in expression and/or in activity level or in prevalence and/or incidence.
  • determining can refer to measuring or ascertaining the quantity or amount of anti-coronavirus or anti -respiratory virus antibody levels in a sample.
  • Methods and techniques used to determining an activity or an event or a quantity or an amount or a change in expression and/or in activity level or in prevalence and/or incidence as used herein can refer to the steps that the skilled person would take to measure or ascertain some quantifiable value.
  • the art is familiar with the ways to measure an activity or an event or a quantity or an amount or a change in expression and/or in activity level or in prevalence and/or incidence.
  • contacting can refer to the placement in direct physical association; includes both in solid and liquid form. “Contacting” is often used interchangeably with “exposed.” In some aspects, “contacting” refers to incubating a molecule (such as an antibody) with a biological sample.
  • control can refer to a reference standard, for example, a positive control or negative control.
  • a positive control is known to provide a positive test result.
  • a negative control is known to provide a negative test result.
  • the reference standard can be a theoretical or computed result, for example a result obtained in a population.
  • Described herein are methods (e.g., multiplex bead-based immunoassays) that are sensitive and specific for the detection of immunoglobulin G (IgG), IgM, and IgA antibodies (Abs) for SARS-CoV-2 infection using noninvasive oral fluid (OF, also referred to herein as saliva or oral fluid sample) specimens.
  • the methods disclosed herein are practical and non-invasive that can be used for large-scale monitoring of seroprevalence of SARS-CoV-2 infections and evaluation of the magnitude and duration of antibody responses after infection, to improve interventions to curb community transmission.
  • Non-invasive oral fluid samples e.g., saliva samples
  • saliva samples which can be self-collected at home and mailed or dropped off at a lab for testing can overcome these barriers and fill- in important herd immunity knowledge gaps.
  • oral fluid samples such as saliva samples that are collected at home can avoid exposure to health care workers who are required to take blood samples as well as eliminate the exposure risk to the patient by avoiding a visit to a testing facility or a physician’s office.
  • SARS-CoV-2 As information emerges about the duration of antibody responses to SARS-CoV-2, it may be important to repeatedly monitor antibody titers over time until surrogates of protection are known, thus collecting oral fluid sample has certain advantages.
  • Saliva for example, can be self-collected. Furthermore, saliva as well as other oral fluid samples can be stored at ambient temperatures for short periods of time ( ⁇ 24 hours) prior to processing and storing. These characteristics of saliva collection can allow large scale saliva-based serosurveillance for SARS-CoV-2 infection, via self-collection and mail-in, as has been done for hepatitis A virus in the United Kingdom (Morris-Cunnington et ak, American Journal of Epidemiology, Vol. 159, Issue 8, April 15, 2004, pp. 786-794).
  • Oral fluid sample based serological methods have several limitations compared to blood-based methods.
  • diagnostic accuracy of oral fluid-based serology is typically lower than that of blood-based methods but the diagnostic accuracy depends on the antigen, assay type, and quality of the collection and testing procedures. Because antibody levels in oral fluids are typically lower than levels observed in sera or plasma, the sensitivity of oral fluid-based testing may be impaired.
  • antigens can be identified that perform with optimized sensitivity and/or specificity in, for example, saliva.
  • saliva for example, has high background mean fluorescence intensity (MFI) due to non-specific binding of salivary macromolecules and exogenous particles to beads which can lead to inflated MFI signals and miss- classification of negatives as positives.
  • control beads can be added into each of the multiplex assays to appropriately adjust each antigen-specific MFI to account for background MFI signal in oral fluid samples, such as saliva.
  • the methods disclosed herein can be used to identify prior SARS-CoV-2 infection using an oral fluid sample (e.g. saliva) obtained from a subject.
  • Disclosed herein are multiplex assay methods that can be used to detect one or more pathogenic specific antibodies in an oral fluid sample.
  • methods for detecting one or more anti-coronavirus antibodies in an oral fluid sample are methods for detecting one or more anti-coronavirus antibodies in an oral fluid sample, the method comprising: a) obtaining or having obtained an oral fluid sample from a subject, wherein the oral fluid sample comprises one or more anti-coronavirus antibodies; b) contacting the oral fluid sample with one or more coronavirus antigens under conditions where the one or more coronavirus antigens bind to the one or more anti-coronavirus antibodies to form a detectable complex, wherein each of the one or more antigens are covalently coupled to a bead comprising a detectable label; and c) detecting the presence of the detectable complexes; thereby detecting the presence of the one or more anti-coronavirus antibodies in the oral fluid sample.
  • the methods can comprise obtaining or having obtained an oral fluid sample from a subject.
  • the oral fluid sample can comprise one or more anti-coronavirus antibodies.
  • the oral fluid sample can comprise one or more classes of anti-coronavirus antibodies.
  • the methods can comprise contacting the oral fluid sample with one or more coronavirus antigens under conditions where the one or more coronavirus antigens bind to the one or more anti- coronavirus antibodies to form a detectable complex.
  • each of the one or more antigens can be covalently coupled to a bead comprising a detectable label.
  • the methods can comprise detecting the presence of the detectable complexes.
  • the methods can comprise detecting the presence of the detectable complexes, thereby detecting the presence of the one or more anti-coronavirus antibodies in the oral fluid sample.
  • the anti-coronavirus antibodies can be specific to one or more SARS-CoV-2 antigens.
  • methods for detecting one or more anti-respiratory virus antibodies in an oral fluid sample comprising: a) obtaining or having obtained an oral fluid sample from a subject, wherein the oral fluid sample comprises one or more anti- respiratory virus antibodies; b) contacting the oral fluid sample with one or more respiratory virus antigens under conditions where the one or more respiratory virus antigens bind to the one or more anti-respiratory virus antibodies to form a detectable complex, wherein each of the one or more antigens are covalently coupled to a bead comprising a detectable label; and c) detecting the presence of the detectable complexes; thereby detecting the presence of one or more anti-respiratory virus antibodies in the oral fluid sample.
  • Also disclosed herein are methods for detecting a respiratory virus in an oral fluid sample comprising: a) obtaining or having obtained the oral fluid sample from a subject, wherein the oral fluid sample comprises one or more respiratory viruses; b) contacting the oral fluid sample with one or more antibodies under conditions where the one or more antibodies bind to the one or more respiratory viruses to form a detectable complex, wherein each of the one or more antibodies are covalently coupled to a bead comprising a detectable label; and c) detecting the presence of the detectable complexes; thereby detecting the presence of the respiratory virus in the oral fluid sample.
  • the methods can comprise obtaining or having obtained an oral fluid sample from a subject.
  • the oral fluid sample can comprise one or more anti- respiratory virus antibodies.
  • the methods can comprise contacting the oral fluid sample with one or more respiratory virus antigens under conditions where the one or more respiratory virus antigens bind to the one or more anti-respiratory virus antibodies to form a detectable complex.
  • each of the one or more respiratory virus antigens can be covalently coupled to a bead comprising a detectable label.
  • the methods can comprise detecting the presence of the detectable complexes.
  • the methods can comprise detecting the presence of the detectable complexes, thereby detecting the presence of the one or more anti-respiratory virus antibodies in the oral fluid sample.
  • the one or more one or more anti-respiratory virus antibodies can be an anti-coronavirus antibody.
  • the one or more respiratory virus antigens can be a coronavirus antigen.
  • the anti-coronavirus antibodies can be specific to one or more SARS-CoV-2 antigens.
  • the classes of anti-coronavirus antibodies can immunoglobulin G (IgG), immunoglobulin A (IgA) or immunoglobulin M (IgM).
  • the classes of anti-coronavirus antibodies can be subtypes of IgG. Examples of IgG subtypes are IgGl, IgG2, IgG3 and IgG4.
  • the one or more anti-coronavirus antibodies can be an IgG, IgA or IgM antibody.
  • the one or more anti-respiratory virus antibodies can be an IgG, IgA or IgM antibody.
  • IgG and IgA can provide information regarding infection post 10-14 days.
  • IgG or IgA levels can be sustained for many months can be an indication of a prior viral exposure and/or vaccination which can indicate (future) protection to a secondary infection.
  • IgM can sometimes elevate sooner than IgG and IgA and can wane within about 30 days, and thus can indicate a recent infection and providing information regarding outbreak situations.
  • the anti-respiratory virus antibodies can be specific to an influenza A antigen, an influenza B antigen, a Parainfluenza antigen, a respiratory syncytial virus (RSV) antigen, a severe acute respiratory syndrome (SARS) antigen, a middle east respiratory syndrome (MERS) antigen, ahCoVs 229e antigen, aNL63 antigen, aHKUl antigen, an OC43 antigen, an adenovirus antigen, rhinovirus antigen, an enterovirus antigen, a SARS- CoV-2 antigen, or a combination thereof.
  • RSV respiratory syncytial virus
  • SARS severe acute respiratory syndrome
  • MERS middle east respiratory syndrome
  • ahCoVs 229e antigen ahCoVs 229e antigen
  • aNL63 antigen a middle east respiratory syndrome
  • aHKUl antigen an OC43 antigen
  • an adenovirus antigen adenovirus antigen
  • rhinovirus antigen an enter
  • the one or more coronavirus antigens can comprise a coronavirus nucleocapsid protein, a spike (S) protein, a receptor binding domain, the entire S protein ectodomain, or an epitope containing a fragment thereof.
  • the one or more coronavirus antigens can comprise all or an epitope containing a polypeptide fragment of a coronavirus nucleocapsid protein, a spike (S) protein, a receptor binding domain, or the entire S protein ectodomain.
  • the S protein can comprise a SI domain and a S2 domain.
  • the one or more coronavirus antigens can be one or more of the coronavirus antigens provided in Table 1 or a fragment thereof.
  • the coronavirus antigen or the anti -respiratory virus antigen can be an anti-ectodomain (ECD) IgG antigen.
  • the one or more anti-respiratory virus antigens can comprise a coronavirus nucleocapsid protein, a spike (S) protein, a receptor binding domain, the entire S protein ectodomain, or an epitope containing a fragment thereof.
  • the one or more anti-respiratory virus antigens can comprise all or an epitope containing a polypeptide fragment of a coronavirus nucleocapsid protein, a spike (S) protein, a receptor binding domain, or the entire S protein ectodomain.
  • the S protein can comprise a SI domain and a S2 domain.
  • the one or more coronavirus antigens can be one or more of the coronavirus antigens provided in Table 1 or a fragment thereof. In some aspects, the one or more anti-respiratory virus antigens can be one or more of the anti- respiratory virus antigens provided in Table 1 or a fragment thereof.
  • any of the antigens used in any of the methods disclosed herein can be or comprise an epitope of the viral (e.g., a coronavirus or a respiratory virus) antigen.
  • the antigens can be selected to comprise one or more epitopes or two or more viral antigens.
  • two or more epitopes can be from the same or different coronavirus (or anti-respiratory virus).
  • two or more viral antigens can be from the same or different proteins from the same or different coronaviruses (or anti- respiratory virus).
  • two or more epitopes from the same or different viral antigen can be different.
  • a combination of viral antigens can be used from different virus classes or virus families.
  • the one or more antigens can be one or more of the antigens provided in Table 1 or a fragment thereof.
  • the methods disclosed herein can apply one or more of the cut-off values in Table 5. In some aspects, the methods disclosed herein can further comprise applying one or more of the cut-off values in Table 5 to the corresponding antigen used. In some aspects, the methods disclosed herein can further comprise normalizing with background data. In some aspects, the methods disclosed herein can further comprise subtracting the background data. In some aspects, any of the algorithms disclosed herein can include subtracting the BSA signal (non-specific binding of antibodies to blocked, non antigen supports [beads]) from each sample’s antigen-specific data. In some aspects, the algorithm can be one or more of the algorithms recited in Table 8. In some aspects, the algorithm can be one or more of the algorithms disclosed in Example 2.
  • the methods disclosed herein can further comprise the use of any of the algorithms described in Example 2 to impart improved sensitivity and specificity of the assay when compared to the performance of an assay with a single viral protein by combining the analysis of data from multiple bead sets containing different viral antigens.
  • any of the algorithms disclosed herein can include determining the ratio(s) of epitope/antigen-specific data divided by the background (BSA) data or antigen-specific data divided by total antibody data for each sample.
  • BSA background
  • total antibody data can be generated with commercially available tests or by including a reporter bead (with, for example, an anti-IgG antibody to capture total IgG) into the multiplex test.
  • the algorithms used herein including BSA subtracted (or normalized) data can result in 100% sensitivity and 100% specificity.
  • cutoff values that discriminate positive samples from negative samples can be calculated using either background subtracted data or raw data. For example, when the cutoff is calculated using background subtracted data (see, for example, Table 5) then the background of each sample needs to be subtracted accordingly prior to applying the cutoff. When a different cutoff value is used without subtracting the background first, the cutoff values can be applied to the raw data.
  • the methods disclosed herein can further comprise setting a cutoff based on a receiver operating characteristic-optimized MFI cutoff for each anti-coronavirus antibody or anti-respiratory virus antibody.
  • a respiratory virus in an oral fluid sample comprising: a) obtaining or having obtained the oral fluid sample from a subject, wherein the oral fluid sample comprises one or more respiratory viruses; b) contacting the oral fluid sample with one or more antibodies under conditions where the one or more antibodies bind to the one or more respiratory viruses to form a detectable complex, wherein each of the one or more antibodies are covalently coupled to a bead comprising a detectable label; and c) detecting the presence of the detectable complexes; thereby detecting the presence of the respiratory virus in the oral fluid sample.
  • the methods can comprise obtaining or having obtained an oral fluid sample from a subject.
  • the oral fluid sample can comprise one or more anti -respiratory viruses.
  • the methods can comprise contacting the oral fluid sample with one or more antibodies under conditions where the one or more antibodies bind to the one or more respiratory viruses to form a detectable complex.
  • each of the one or more antibodies can be covalently coupled to a bead comprising a detectable label.
  • the methods can comprise detecting the presence of the detectable complexes.
  • the methods can comprise detecting the presence of the detectable complexes, thereby detecting the presence of the respiratory virus in the oral fluid sample.
  • one or more antibodies can be derived from the same or different hosts.
  • methods for detecting one or more viral polypeptides in an oral fluid sample comprising: a) obtaining or having obtained an oral fluid sample from a subject, wherein the oral fluid sample comprises the one or more viral polypeptides; b) contacting the oral fluid sample with one or more antibodies under conditions where the one or more antibodies bind to the one or more viral polypeptides to form a detectable complex, wherein each of the one or more antibodies are covalently coupled to a bead comprising a detectable label; and c) detecting the presence of the detectable complexes; thereby detecting the presence of the one or more viral polypeptides in the oral fluid sample.
  • the methods can comprise obtaining or having obtained an oral fluid sample from a subject.
  • the oral fluid sample can comprise one or more viral polypeptides.
  • the methods can comprise contacting the oral fluid sample with one or more antibodies under conditions where the one or more antibodies bind to the one or more viral polypeptides to form a detectable complex.
  • each of the one or more antibodies can be covalently coupled to a bead comprising a detectable label.
  • the methods can comprise detecting the presence of the detectable complexes.
  • the methods can comprise detecting the presence of the detectable complexes, thereby detecting the presence of the viral polypeptide in the oral fluid sample.
  • one or more antibodies can be derived from the same or different hosts.
  • the viral polypeptide can be respiratory virus.
  • the viral polypeptide can be derived from or a fragment from a respiratory virus.
  • the methods disclosed herein can be used for the diagnosis, prognosis, risk assessment, risk stratification, therapy control and/or post-operative control of a disorder or medical condition in the subject.
  • methods for diagnosing, prognosing, assessing risk, risk stratification, therapy control and/or post-operative control of a disorder or medical condition in the subject comprising: detecting one or more anti-virus antibodies in an oral fluid sample, the method comprising: a) obtaining or having obtained an oral fluid sample from a subject, wherein the oral fluid sample comprises one or more anti-virus antibodies; b) contacting the oral fluid sample with one or more virus antigens under conditions where the one or more virus antigens bind to the one or more anti virus antibodies to form a detectable complex, wherein each of the one or more virus antigens are covalently coupled to a bead comprising a detectable label; and c) detecting the presence of the detectable complexes; thereby detecting the presence of the one or more anti-virus antibodies in
  • the anti-virus antibodies can be anti- coronavirus antibodies, anti-respiratory virus antibodies, and the like.
  • the disorder or medical condition can be COVID-19.
  • the disorder or medical condition can be acute respiratory infection, bronchiolitis, common cold, croup, influenza- like-illness, or pneumonia.
  • the disorder or medical condition can be COVID- 19, acute respiratory infection, bronchiolitis, common cold, croup, influenza-like-illness, pneumonia or a combination thereof.
  • the subject can be a human subject.
  • the sample can be from a subject exposed to or suspected of being exposed to a coronavirus.
  • the sample can be from a subject not exposed to or not suspected of being exposed to a coronavirus.
  • the sample can be from a subject exposed to or suspected of being exposed to a respiratory virus.
  • the sample can be from a subject not exposed to or not suspected of being exposed to a respiratory virus.
  • the methods disclosed herein can be performed using a sample from a subject that has an active infection.
  • the methods disclosed herein can be performed using a sample from a subject that has a prior infection.
  • the coronavirus can be severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), severe acute respiratory syndrome (SARS), middle east respiratory syndrome (MERS), human coronavirus 229E, human coronavirus NL63, Miniopterus bat coronavirus 1 ,Miniopterus bat coronavirus HKU8, porcine epidemic diarrhea virus, Rhinolophus bat coronavirus HKU2, Scotophilus bat coronavirus 512, bovine coronavirus, human coronavirus OC43, human coronavirus HKU1, murine coronavirus, Pipistrellus bat coronavirus HKU5, Rousettus bat coronavirus HKU9, Tylonycteris bat coronavirus HKU4, hedgehog coronavirus 1, infectious bronchitis virus, beluga whale coronavirus SW1, infectious bronchitis virus, Bulbul coronavirus HKU11, pangolin coronavirus, porcine coronavirus HKU15, WIVl-CoV
  • the respiratory virus can be a coronavirus, an influenza A virus, an influenza B virus, a Parainfluenza virus, a respiratory syncytial virus (RSV), a severe acute respiratory coronavirus syndrome-CoV (SARS-CoV), a middle east respiratory syndrome coronavirus (MERS-CoV), a human coronavirus (hCoV) 229e, a human coronavirus NL63, a human coronavirus HKU1, an human coronavirus OC43, an adenovirus, a rhinovirus, an enterovirus, a SARS-CoV-2, or a combination thereof.
  • RSV respiratory syncytial virus
  • SARS-CoV severe acute respiratory coronavirus syndrome-CoV
  • MERS-CoV middle east respiratory syndrome coronavirus
  • hCoV human coronavirus 229e
  • a human coronavirus NL63 a human coronavirus HKU1
  • an human coronavirus OC43 an
  • Coronaviruses are a group of RNA viruses that can cause respiratory tract infections that can range from mild to lethal. Mild illnesses include the common cold. Lethal illnesses can cause SARS, MERS and COVID-19. Coronaviruses constitute the subfamily Orthocoronavirinae, in the family Coronaviridae, order Nidovirales, and realm Riboviria. They are enveloped viruses with a positive-sense single-stranded RNA genome and a nucleocapsid of helical symmetry that is wrapped in an icosahedral protein shell. The genome size of coronaviruses ranges from approximately 26 to 32 kilobases. Club-shaped spikes that project from their surface are characteristic of coronaviruses.
  • the methods disclosed herein can further comprise a negative control. In some aspects, the methods disclosed herein can further comprise performing the any of the methods described herein in parallel with a negative control.
  • the negative control can comprise a sample from a subject not exposed to a coronavirus. In some aspects, the negative control can comprise a sample from a subject not exposed to a respiratory virus. In some aspects, the negative control can be performed to be used to compare the signal from saliva samples collected from a subject with a prior SARS-CoV-2 infection to that of negatives. In some aspects, the negative control can be used to generate cut off values to classify saliva samples as positive or negative for SARS-CoV-2 antibodies.
  • a positive test can be the result of at least two positive signals from two different antigens.
  • the IgG signal can be higher than that observed among negative samples.
  • the IgG signal must be higher than that observed among negative samples.
  • the step of detecting can comprise detecting by microarray. In some aspects, the step of detecting can comprise detecting by ELISA.
  • the sensitivity of the methods for detecting one or more antibodies in oral fluid samples can be equivalent or increased compared to detecting analogous antibodies or virus-specific antibodies in a blood sample.
  • the specificity of the methods for detecting one or more antibodies in oral fluid samples can be equivalent or increased compared to detecting analogous antibodies or virus-specific antibodies in a blood sample.
  • the sensitivity of the methods for detecting a respiratory virus in oral fluid samples can be equivalent or increased compared to detecting a viral antigen in a blood sample.
  • the sensitivity and specificity using an oral fluid sample can be equivalent to the results from a blood sample.
  • the sensitivity and specificity using an oral fluid sample can be equivalent to using RT-PCR.
  • the beads are in a solution.
  • the beads can comprise a detectable label or detection tag (e.g., FLAGTM tag, epitope or protein tags, such as myc tag,
  • the detectable label or detection tag can be a protein purification affinity tag.
  • Epitope tags are short stretches of amino acids to which a specific antibody can be raised, which in some aspects allows one to specifically identify and track the tagged protein that has been added to a substrate.
  • Beads as disclosed herein are commercially available.
  • An example of beads that can be useful in the methods disclosed herein include MagPlex-TAGTM microspheres.
  • Detection of the tagged molecule can be achieved using a number of different techniques. Examples of such techniques include: immunohistochemistry, immunoprecipitation, flow cytometry, immunofluorescence microscopy, ELISA, immunoblotting (“Western blotting”), and affinity chromatography.
  • Epitope tags add a known epitope (e.g., antibody binding site) on the subject protein, to provide binding of a known and often high-affinity antibody, and thereby allowing one to specifically identify and track the tagged protein.
  • epitope tags include, but are not limited to, myc, T7, GST, GFP, HA (hemagglutinin), V5 and FLAG tags. The first four examples are epitopes derived from existing molecules.
  • FLAG is a synthetic epitope tag designed for high antigenicity (see, e.g., U.S. Pat. Nos. 4,703,004 and 4,851,341).
  • Epitope tags can have one or more additional functions, beyond recognition by an antibody.
  • label, detection tag, epitope-tag, affinity tag or protein purification affinity tag can be His-tag, a FLAG-tag, a HA (hemagglutinin)-tag, a Strep-tag, a C9-tag, a glutathione S-transferase tag, a maltose-binding protein tag, a T7 tag, a V5 tag, an S tag, a SUMO tag, a TAP tag, a TRX tag, a calmodulin binding peptide, a chitin binding domain, a E2 epitope, a HaloTag, a HSV tag, a HBH tag, a KT3 tag, VSV-G tag, CD tag, Avitag, or GFP-tag or a myc-tag.
  • the sequences of these tags are described in the literature and well known to the person of skill in art.
  • the methods disclosed herein comprise the step of contacting the oral fluid sample with one or more antibodies or a plurality of antibodies under conditions where the one or more antibodies or a plurality of antibodies bind to the one or more respiratory viruses to form a detectable complex.
  • the antibody is capable of specifically binding to the epitope of the respiratory virus (e.g., a coronavirus).
  • the antibody can be a monoclonal antibody. In some aspects, the antibody can be a polyclonal antibody.
  • the methods disclosed herein comprise the step of contacting the oral fluid sample with one or more respiratory virus antigens or a plurality of respiratory virus antigens that are capable of specifically binding to the one or more (or a plurality ol) anti- respiratory virus antibodies (e.g., anti-coronavirus antibodies), thereby forming a detectable complex.
  • one or more respiratory virus antigens or a plurality of respiratory virus antigens that are capable of specifically binding to the one or more (or a plurality ol) anti- respiratory virus antibodies (e.g., anti-coronavirus antibodies), thereby forming a detectable complex.
  • the methods disclosed herein can further comprise determining the anti-coronavirus antibody level in the oral fluid sample. In some aspects, the methods disclosed herein can further comprise determining the anti-respiratory virus antibody level in the sample.
  • the methods disclosed herein can further comprise determining anti- coronavirus antibody titers or anti-respiratory virus antibody titers.
  • a serial dilution of a known antibody (e.g., standard) specific to each SARS-CoV-2 or respiratory virus antigen can be incubated and tested using the methods disclosed herein to generate a standard curve for each SARS-CoV-2 or respiratory virus antibody. The signal of each test can then be compared to the standard curve for each coronavirus or respiratory-virus antigen contained in the method (e.g., multiplex) to determine the anti-coronavirus or anti-respiratory virus antibody titer.
  • the oral fluid sample can be whole saliva, gingival crevicular fluid, oral mucosal transudates, or serum enriched salivary.
  • the oral fluid sample can comprise one or more anti-coronavirus antibodies.
  • the oral fluid sample can comprise one or more classes of anti-coronavirus antibodies.
  • the oral fluid sample can be collected around or after 20 minutes after eating. For example, if the subject has eaten within 20 minutes prior to collecting or obtaining a sample, the sample can be collected or obtained by waiting to until the 20 minute interval has passed.
  • the oral fluid sample can be obtained using a commercially available saliva collection device (e.g., Oracol SI 0 or S 14 available from Malvern Medical Developments).
  • the saliva collection device can be an oral swab device.
  • the oral swab device can comprise a sponge.
  • An oral fluid sample can be obtained from a subject by inserting, for example, the sponge component between a subject’s gums and cheeks at the gum/tooth line and rubbing the sponge against the gums for at least one minute (e.g., similar to brushing their gum/teeth line). The sponge should be completely saturated.
  • Oral fluid samples can be refrigerated at 2°C - 8°C on ice packs until processing.
  • the processing of the oral fluid sample can be performed by centrifuging the sample, for example, at 1500 g (> 1000 g) at 4°C for about 10 min (centrifugation at room temperature can be done if a refrigerated centrifuge is not available). Oral fluid samples can be placed in storage boxes at -70°C until use.
  • the oral fluid sample can be a serum enriched sample.
  • the serum enriched sample can be obtained by rubbing or brushing the gums/tooth line with a device (e.g., a toothbrush) and collecting passive drool at a designated time after brushing.
  • the saliva is enriched with serum components, including, for example, antibodies, in part due to microabrasions along the gum line.
  • the saliva sample can be obtained by rubbing or brushing the gums/tooth line with a device comprising a sponge that collects (e.g., absorbs) the oral fluid into the sponge, and depositing a passive drool sample comprising the serum enriched saliva into the collection device along with the device comprising the sponge immediately following the brushing of the gums/tooth line, together into the collection device, thus collecting both oral fluid in the sponge and serum enriched saliva as passive drool, in the collection device.
  • a device comprising a sponge that collects (e.g., absorbs) the oral fluid into the sponge
  • a passive drool sample comprising the serum enriched saliva into the collection device along with the device comprising the sponge immediately following the brushing of the gums/tooth line, together into the collection device, thus collecting both oral fluid in the sponge and serum enriched saliva as passive drool, in the collection device.
  • the oral fluid sample can be about 5 pL, 50 pL, 100 pL, 500 pL, 750 pi, or more, or any amount in between. In some aspects, the oral fluid sample can be about 1000 pL. In some aspects, the method disclosed herein requires a minimal volume requirement. The use of the multiplex methods disclosed herein allows for lower volume requirements as compared to traditional ELISAs.
  • the methods disclosed herein can further comprise obtaining or having obtained a second oral fluid sample from a subject at a different time. In some aspects, the methods disclosed herein can further comprise obtaining or having obtained a second oral fluid sample from a subject at a different time and repeating the contacting step and the detection steps as described in the methods disclosed herein.
  • the methods for detecting a respiratory virus comprising the step of contacting the oral fluid sample with one or more antibodies, the one or more antibodies can be commercially available.
  • the antibody can be an antibody fragment or a biologically active variant thereof.
  • monoclonal antibodies can be made by recombinant DNA. DNA encoding monoclonal antibodies can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). Libraries of antibodies or active antibody fragments can also be generated and screened using phage display techniques.
  • antibody fragments can be produced through enzymatic treatment of a full-length antibody. Digestion of antibodies to produce fragments thereof, particularly, Fab fragments, can be accomplished using routine techniques known in the art. For instance, digestion can be performed using papain. Papain digestion of antibodies typically produces two identical antigen binding fragments, called Fab fragments, each with a single antigen binding site, and a residual Fc fragment. Pepsin treatment yields a fragment that has two antigen combining sites and is still capable of cross-linking antigen. Antibodies incorporated into the present bi- functional allosteric protein-drug molecules can be generated by digestion with these enzymes or produced by other methods.
  • the fragments can also include insertions, deletions, substitutions, or other selected modifications of particular regions or specific amino acids residues, provided the activity of the antibody or antibody fragment is not significantly altered or impaired compared to the non-modified antibody or antibody fragment. These modifications can provide for some additional property, such as to remove/add amino acids capable of disulfide bonding, to increase its bio-longevity, to alter its secretory characteristics, etc. In any case, the antibody or antibody fragment must possess a bioactive property, such as specific binding to its cognate antigen. Functional or active regions of the antibody or antibody fragment can be identified by mutagenesis of a specific region of the protein, followed by expression and testing of the expressed polypeptide. Such methods are readily apparent to a skilled practitioner in the art and can include site-specific mutagenesis of the nucleic acid encoding the antibody or antibody fragment.
  • antibody can also refer to a human antibody and/or a humanized antibody.
  • Many non-human antibodies e.g., those derived from mice, rats, or rabbits
  • are naturally antigenic in humans and thus can give rise to undesirable immune responses when administered to humans. Therefore, the use of human or humanized antibodies in the methods serves to lessen the chance that an antibody administered to a human will evoke an undesirable immune response.
  • Antibody humanization techniques generally involve the use of recombinant DNA technology to manipulate the DNA sequence encoding one or more polypeptide chains of an antibody molecule.
  • a humanized form of a non-human antibody is a chimeric antibody or antibody chain (or a fragment thereof, such as an Fv, Fab, Fab', or other antigen binding portion of an antibody) which contains a portion of an antigen binding site from a non-human (donor) antibody integrated into the framework of a human (recipient) antibody.
  • the Fv region is a minimal fragment containing a complete antigen-recognition and binding site consisting of one heavy chain and one light chain variable domain.
  • the three CDRs of each variable domain interact to define an antigen-biding site on the surface of the Vh-Vl dimer.
  • the six CDRs confer antigen-binding specificity to the antibody.
  • a “single-chain” antibody or “scFv” fragment is a single chain Fv variant formed when the VH and VL domains of an antibody are included in a single polypeptide chain that recognizes and binds an antigen.
  • single-chain antibodies include a polypeptide linker between the Vh and VI domains that enables the scFv to form a desired three-dimensional structure for antigen binding.
  • the antibody can be a single chain variable fragment (scFv), a camelid antibody, a nanobody, or a shark vNAR antibody.
  • scFv single chain variable fragment
  • Various combinations of individual pairs of antibodies to antigens (or virus to antibody) are encompassed by the present disclosure. Each combination, including a specific anti-coronavirus antibody and its corresponding coronavirus antigen (making up a pair or when bound producing a detectable complex) can be selected independently.
  • the component parts need to be associated in a compatible manner. By combining two or more different individual pairs in any of the methods disclosed herein, the methods can be used to distinguish between two or more viruses that share common symptoms.
  • the methods can be performed using a plurality (e.g., two or more) of antigens to distinguish between two or more anti-viral antibodies (e.g., two different coronaviruses or SARS-CoV-2 vs influenza A), and thus two more viruses that share common symptoms.
  • a plurality e.g., two or more of antigens to distinguish between two or more anti-viral antibodies
  • the specificity and sensitivity of the methods can be increased or enhanced.
  • two different antigens to the same antibody e.g., same coronavirus
  • a plurality of viral proteins or antibodies capable of binding to one or more viral antigens can be each be coupled to beads thereby forming a particular combination (e.g., a detectable complex), such that the combination selected can increase the sensitivity and specificity of the detection.
  • the methods can be performed using a plurality (e.g., two or more) of coronavirus antigens or respiratory virus antigens to distinguish between two or more viruses that share common symptoms.
  • the methods can be performed using a plurality (e.g., two or more) of antibodies to distinguish between two or more viruses that share common symptoms.
  • Serum and saliva immunoassays to assess COVID-19 infection status Disclosed herein are COVID-19 multiplex antibody assays.
  • the methods (e.g., immunoassays) described herein can be based on Luminex technology.
  • Luminex technology For both serum or plasma-based as well as an oral fluid based (e.g.
  • SARS-CoV-2 antigens and antigens from other respiratory pathogens can be covalently coupled to, for example, MagPlex magnetic microspheres (Luminex) (also referred to herein as “beads” or “magnetic beads”).
  • Luminex MagPlex magnetic microspheres
  • the magnetic beads can serve as a solid phase and can react with the serum or saliva sample.
  • Pathogen-specific antibodies in the sample can bind to the immobilized antigens and be detected using fluorophore-labeled Ig class-specific antibodies (e.g., fluorophore-labeled anti -human IgG, anti -human IgM or anti -human IgA antibody).
  • fluorophore-labeled Ig class-specific antibodies e.g., fluorophore-labeled anti -human IgG, anti -human IgM or anti -human IgA antibody.
  • Antibody assay - Coupling of antigens to magnetic beads can be from several sources including commercial entities and academic research labs, e.g., The Native Antigen Company, GenScript and Sino Biological provide several high-quality antigens that can be used in the methods. RBD antigens can also be used. Prior to coupling the antigens to the beads, the antigens may need to be purified by size exclusion chromatography to remove any amine-containing substances that may interfere with the carbodiimide coupling reaction (e.g., Tris, urea) and eluted in phosphate buffer saline (PBS, pH 7.4) or another amine-free buffer that is compatible with the antigen.
  • PBS phosphate buffer saline
  • Antigens are covalently coupled to, for example, carboxylated magnetic MagPlex Microspheres (Luminex Corp., Austin, TX), according to the Luminex xMAP Cookbook 2 nd edition with the following modifications: 1) After activation with N- hydroxysulfosuccinimide sodium salt (Sulfo-NHS) and l-ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDC), the beads were washed once (instead of twice) with 250 pL 50 mM 2-morpholinoethanesulfonic acid (MES) buffer, pH 5.0; 2) without soni cation (instead of with); and 3) antigens are diluted in 400 pL PBS (instead of MES) prior to addition to the activated beads.
  • MES 2-morpholinoethanesulfonic acid
  • Beads are considered activated following treatment, for example, with 10 pi of 50 mg/ml Sulfo-N-hydroxysuccinimide (Sulfo-NHS) and 10 pi of 50 mg/ml l-ethyl-3-[3-dimethylaminopropyl]carbodiimide (EDC) for 20 minutes at room temperature.
  • Sulfo-NHS Sulfo-N-hydroxysuccinimide
  • EDC dimethylaminopropyl]carbodiimide
  • the coupling of antigens to the magnetic beads can be confirmed using either a serum sample containing pathogen-specific antibodies or using commercially available antibodies against the antigen or antibodies against any antigen tag if present (e.g., anti-His- tag antibody) followed by a secondary R-phycoerythrin (PE) labeled antibody specific to the primary host antibody (e.g., anti-mouse PE antibody, Jackson ImmunoResearch Laboratories Inc., West Grove, PA).
  • a serum sample containing pathogen-specific antibodies or using commercially available antibodies against the antigen or antibodies against any antigen tag if present (e.g., anti-His- tag antibody) followed by a secondary R-phycoerythrin (PE) labeled antibody specific to the primary host antibody (e.g., anti-mouse PE antibody, Jackson ImmunoResearch Laboratories Inc., West Grove, PA).
  • PE R-phycoerythrin
  • Antibody assay -Multiplex serum/plasma assay Serum or plasma samples can be tested for pathogen-specific IgG, IgM and IgA antibodies by a multiplex assay that measures the immune response to the antigens in the same sample. Up to 50 different antigens can be multiplexed. Antigen-coupled beads can be vortexed, sonicated and diluted in assay buffer (PBS with 0.05% Tween20 and 1% bovine serum albumin, hereafter “buffer”).
  • assay buffer PBS with 0.05% Tween20 and 1% bovine serum albumin, hereafter “buffer”.
  • 40 pL of the bead mix (1,500 beads of each set) and 10 pL of the serum sample can be diluted 1 :200 in buffer and then added to each well of a microtiter plate (final serum/plasma dilution: 1:1,000).
  • the plate is then covered incubated for 1 h on a plate shaker at 500 rpm.
  • the beads are washed at least twice with 200 pL PBST using either an automated magnetic bead wash station (Bio-Plex Pro Wash Station, Bio-Rad, Hercules, CA) or a handheld magnetic plate separator.
  • PE-labeled anti-human IgG or anti-human IgA or IgM (or anti-IgGl or IgG3) antibody can be diluted 1:100 in buffer and added to each well.
  • the plate is incubated for lh as described herein.
  • the magnetic beads can be washed once more, suspended in 100 pL assay buffer and the fluorescence signal can be measured on a Bio-Plex 200 instrument (Bio- Rad, Hercules, CA) or MagPIX instrument (Luminex).
  • Antibody assay -Multiplex oral fluid assay Saliva samples can be centrifuged for 5 min at 20,000 g to remove any debris and precipitated mucins. Undiluted supernatants were then tested for anti-respiratory virus antibodies using the procedure described herein. Each well contained 40 pL bead mix and 10 pL saliva; the final saliva dilution was 1:5 in buffer. The final saliva dilution can be further optimized depending on the target isotype (IgG vs.
  • a set of at least 500 pre-COVID specimens can be tested (e.g., specimens negative for antibodies against SARS-CoV-2) to establish a well-informed cut-off.
  • the cutoff can be calculated using the average median fluorescence intensity (MFI), i.e., the read-out signal for each antigen-coupled bead set and adding 3 standard deviations to this mean.
  • MFIs median fluorescence intensity
  • Samples yielding signals (MFIs) that are above the cutoff can be classified as positive for that antigen, and samples with MFIs below the cutoff can be classified as negative for that antigen.
  • Staggered classification Maximum sensitivity followed by maximum specificity.
  • samples can be classified according to their MFI results for the most sensitive antigen in the multiplex (positive if above the threshold) to capture the maximum number of potentially positive samples. This is followed by EXCLUSION of samples with MFIs BELOW the cutoff for a second highly specific antigen.
  • the instances in which a sample is above the cutoff can be counted for each antigen represented in the multiplex assay. For example, in a 12-plex, the maximum score would be 12. In this case, a sample would be classified positive for each antigen represented in the assay. An empiric minimum score can be established to detect true positives and true negatives (e.g., a minimum score of 6 for a 12-plex assay containing 12 SARS-CoV-2 antigen-coupled bead sets).
  • the methods disclosed herein can comprise a positive sample that is positive for at least two antigens representing different epitopes of the virus.
  • SARS-CoV-2 antigen assay Multiplex antigen assay.
  • SARS-CoV-2 multiplex antibody assays Disclosed herein are methods of detecting SARS-CoV-2 in saliva, i.e. test for the presence of the virus rather than antibody against SARS-CoV-2.
  • the beads can be coupled with an anti-SARS-CoV-2 antibody, and then saliva can be added. If the virus is present, it will bind to the bead, and SARS-CoV-2 can be detected using either a PE-labeled anti-SARS-CoV-2 antibody or PE-labeled ACE2 (human receptor to which SARS-CoV-2 binds for cell entry).
  • sandwich antibody assay This type of approach is often referred to as a “sandwich antibody assay”.
  • sandwich approach can also be flipped.
  • an ACE2 can be coupled to the bead and SARS-CoV-2 can be detected using an anti-SARS-CoV 2 antibody.
  • an approach using a sandwich comprising antibodies produced in different hosts can be used.
  • mouse anti- SARS-CoV-2 antibody can be coupled to the beads, and SARS-CoV-2 can then bind to the bead/antibody.
  • rabbit anti-SARS-CoV-2 antibody can be added followed by a PE-labeled anti-rabbit antibody. The R-Phycoerythrin label can serve as the signal for the read-out.
  • kits comprising one or more of the components of the methods described herein.
  • kits comprising one or more coronavirus antigens, one or more respiratory virus antigens, or one or more antibodies for detecting one or more respiratory viruses.
  • the kits can comprise a collection device.
  • the disclosed kits can comprise instructions for preparing oral fluid sample.
  • kits can comprise one or more coronavirus antigens, one or more respiratory virus antigens that comprise coronavirus nucleocapsid protein, a spike (S) protein, wherein the S protein sequence comprises a SI domain and a S2 domain, a receptor binding domain, the entire S protein ectodomain, or an epitope containing fragment thereof or all or an epitope containing fragment of a coronavirus nucleocapsid protein, a spike (S) protein, wherein the S protein sequence comprises a SI domain and a S2 domain, a receptor binding domain, or the entire S protein ectodomain.
  • Example 1 A salivary antibody multiplex panel for accurate identification of SARS-CoV-2 infection
  • Non-invasive SARS-CoV-2 antibody testing is urgently needed to estimate the prevalence of SARS-CoV-2 infection at the population level. Precise knowledge of the local population immunity will allow government bodies to make informed decisions about how and when to loosen stay-at-home directives and to reopen the economy. It was tested whether salivary antibodies to SARS-CoV-2 could serve as a non-invasive alternative to serum SARS-CoV-2 antibodies and could thus provide evidence for prior SARS-CoV-2 infection.
  • the assay s sensitivity and specificity to correctly identify prior SARS-CoV-2 infection based on antigen-specific IgG, IgA and IgM responses in saliva to that in serum were calculated and compared. Matched serum and saliva SARS-CoV-2 antigen-specific IgG responses were strongly correlated.
  • the salivary anti-nucleocapsid (N) protein IgG response resulted in the highest sensitivity for detecting prior SARS-CoV-2 infection (100% sensitivity at >10 days post SARS-CoV-2 symptom onset).
  • the salivary anti-receptor binding domain (RBD) IgG response resulted in 100% specificity.
  • the SARS-CoV-2 pandemic has caused >4.2 million COVID-19 cases and >289 thousand deaths, as of May 12, 2020, involving the populated continents (Dong E, et al. Lancet Infect Dis. 2020 May;20(5):533-4).
  • Development of improved antibody assays to detect historical infection with SARS-CoV-2 is among the top unmet needs in the ongoing pandemic response.
  • GCF gingival crevicular fluid
  • Saliva is more sensitive for SARS-CoV-2 detection in COVID-19 patients than nasopharyngeal swabs. medRxiv. 2020;(2):2020.04.16.20067835), and could thus serve as a biospecimen to measure both presence of the virus by either antigen or RNA detection and antibodies against the virus.
  • Salivary antibody assays can allow individual and population-level assessments of prior SARS-CoV-2 infection at a massive scale. Prior studies have shown that antibodies to SARS-CoV-2 nucleocapsid protein (N), spike protein (S), and the receptor binding domain (RBD) are elevated in serum around 10-18 days after SARS-CoV-2 infection (Long Q-X, et al. Nat Med. 2020 Apr 29; Guo L, et al. Clin Infect Dis. 2020 Mar 21; Zhao J, et al. Clin Infect Dis. 2020 Mar 28; and Okba NMA, et al. Emerg Infect Dis. 2020 Jul;26(7)).
  • N SARS
  • Saliva samples were collected from subjects by gently brushing the gum line with an Oracol S14 Saliva Collection sponge (Malvern Medical, UK) for 60 seconds, or until saturation. This saliva collection method specifically harvests GCF, which is enriched with primarily IgG antibody derived from serum. The sponge was inserted into the storage tube, capped, and stored at 4°C until processing whenever possible. Saliva was separated from the Oracol S14 swabs through centrifugation (10 min at 1500 g) and transferred into the attached 2 mL cryovials. Samples were heat-inactivated at 60°C for 30 minutes prior to analysis.
  • Plasma/serum was also heat inactivated at 60°C for 30 minutes, aliquoted into 2mL cryovials, and stored at ⁇ 20°C until use.
  • SARS-CoV-2 saliva immunoassay Ten SARS-CoV-2 antigens were obtained commercially or from collaborators at Icahn School of Medicine at Mount Sinai (provided in Table 2). This included four SARS-CoV-2 receptor binding domain (RBD) proteins, one ectodomain (ECD) protein containing the SI and S2 subunit of the spike protein, and two nucleocapsid (N) proteins. Each SARS-CoV-2 antigen, along with one SARS-CoV-1 antigen (NAC SARS 2002 N) and one hCoV-229E antigen (Sino Biol.
  • RBD SARS-CoV-2 receptor binding domain
  • ECD ectodomain protein containing the SI and S2 subunit of the spike protein
  • N nucleocapsid
  • hCoV 229E ECD were covalently coupled to magnetic microparticles (MagPlex microspheres, Luminex) (Table 2) (Pisanic N, et al. J Immunol Methods. 2017;448:1-8)).
  • the multiplex panel included a total of 13 bead sets (10 bead sets coupled to SARS-CoV-2 antigens, one coupled to SARS- CoV-1 antigen, one coupled to hCoV-229E antigen, and one control bead coupled to BSA).
  • Saliva samples were centrifuged (5 minutes at 20,000g, 20°C), and 10 pL of oral fluid supernatant was added to 40 pL of assay buffer (phosphate-buffered saline with 0.05% Tween20, 0.02% sodium azide, and 1% BSA) containing 1500 beads of each bead set (Table 2) per microplate well.
  • assay buffer phosphate-buffered saline with 0.05% Tween20, 0.02% sodium azide, and 1% BSA
  • the plate was covered and incubated at room temperature for 1 hour on a plate shaker at 500 rpm. Beads were washed 3 times, 50 pL of PE-labeled anti -human IgG diluted 1 : 100 in assay buffer was added, and the plate was incubated for 1 hour on a plate shaker at 500 rpm.
  • N nucleocapsid protein
  • ECD ectodomain (SI + S2 subunit of spike protein)
  • RBD receptor binding domain
  • the average MFI of the negative saliva samples plus three standard deviations for each antigen-specific IgG, IgA, and IgM were used as MFI cut-offs to classify each saliva and serum sample as positive or negative for prior SARS-CoV-2 infection. Because the true hCoV negative status for saliva and serum samples was unknown, a MFI cut off value was not calculated for anti-Sino Biol. hCoV 229E ECD IgG, IgA, and IgM.
  • Sensitivity and specificity for detecting RT-qPCR confirmed prior SARS-CoV-2 infection was determined for each antigen/isotype pair (IgG, IgM and IgA) in saliva and in serum.
  • Locally weighted regression LOESS was used to visualize and compare the temporal kinetics of saliva and serum antigen-specific IgG, IgA, and IgM responses among individuals with RT-qPCR confirmed prior SARS-CoV-2 infection by days since symptom onset.
  • Saliva and serum samples A total of 33 saliva samples and 206 serum samples were collected from 33 and 196 subjects, respectively, with RT-qPCR confirmed prior SARS-CoV-2 infection. Information on days post symptom onset was collected for each positive participant. A total of 134 saliva samples and 112 serum samples were collected from subjects enrolled in various cohort studies during the year 2016 or prior and were designated as known-negative samples (pre-COVID-19 pandemic) (Table 3).
  • Antigen-specific IgM in matched saliva and serum samples were also significantly correlated for the SARS-CoV-2 and SARS-CoV-1 antigens tested, although the correlation was weaker than antigen-specific IgG in matched saliva and serum samples (Fig. 3).
  • SARS-CoV-2 antigen-specific IgG, IgA, and IgM cut off values.
  • the multiplex immunoassay comprising ten SARS-CoV-2 antigens (2 N proteins, 1 ECD protein, four RBD proteins, two SI subunits, and one S2 subunit), one SARS-CoV-1 antigen (NAC SARS CoV 2002 N), and one hCoV-229E antigen (Sino Biol. hCoV 229E ECD) was used to probe a total of 167 saliva samples from 150 individuals and 324 serum samples from 308 subjects.
  • the range, median, mean, standard deviation, and derived MFI cut off value for each saliva and serum SARS-CoV-2 antigen-specific IgG, IgA, and IgM stratified by negative samples, samples collected ⁇ 10 days post SARS-CoV-2 symptom onset, and >10 days post SARS- CoV-2 symptom onset are provided in Fig. 8 and Fig. 9.
  • Saliva collected at >10 days post symptom onset displayed significantly elevated median MFI compared to negatives for the SARS-CoV-2 antigen-specific IgG tested (Fig. 8).
  • Serum collected at >10 days post symptom onset displayed significantly elevated median MFI compared to negatives for the SARS- CoV-2 antigen-specific IgG, IgA, and IgM tested.
  • the average intra-assay variability (CV%) ranged from 2.66%-17.98%
  • the average inter assay variability (CV%) ranged from 4.60%-27.7% (Table 4).
  • Saliva sensitivity and specificity.
  • SARS-CoV-2 antigen-specific IgG sensitivity ranged from 0.0%-40.0% among saliva samples collected ⁇ 10 days post symptom onset, and 42.9%- 100.0% among saliva samples collected >10 days post symptom onset (Fig. 4).
  • the best sensitivity was achieved by anti-GenScript N IgG, which performed with 100% sensitivity among saliva samples collected >10 days post symptom onset (28/28 individuals with RT-qPCR confirmed prior SARS-CoV-2 infection correctly classified). Specificity ranged from 97.8%- 100.0% for SARS-CoV-2 antigen-specific IgG, with anti-Mt. Sinai RBD IgG performing with 100% specificity for classifying negatives (134/134 negatives correctly classified).
  • IgA Specificity for IgA ranged from 42.3% (anti-GenScript SI IgA) to 100.0% (anti-GenScript SI and anti-NAC S2 IgA), and for IgM ranged from 96.4% [anti-GenScript RBD (i) IgM] to 98.80% (anti-Sino Biol. ECD, anti-GenScript SI, and anti-NAC S2 IgM).
  • Blood sensitivity and specificity.
  • SARS-CoV-2 antigen-specific IgG sensitivity ranged from 1.1 %-26.1 % among serum samples collected ⁇ 10 days post symptom onset, and 23.1%-92.3% among serum samples collected >10 days post symptom onset (Fig. 5).
  • RBD IgG which both performed with 92.3% sensitivity among serum samples collected >10 days post symptom onset (96/104 individuals with RT-qPCR confirmed prior SARS-CoV-2 infection correctly classified). Specificity ranged from 96.4%-99.1% for SARS-CoV-2 antigen-specific IgG, with anti-Mt. Sinai RBD IgG, anti-NAC SI IgG, and anti-NAC S2 IgG, performing with 100% specificity for classifying negatives (112/112 negatives correctly classified).
  • SARS-CoV-2 antigen-specific IgA For SARS-CoV-2 antigen-specific IgA, sensitivity ranged from 0.0%-44.6% among serum samples collected ⁇ 10 days post symptom onset, and 15.4%-95.2% among serum samples collected >10 days post symptom onset (Fig. 5).
  • anti-GenScript RBD IgA performed with the best sensitivity (95.2%) among serum samples collected >10 days post symptom onset (99/104 individuals with RT-qPCR confirmed prior SARS-CoV-2 infection correctly classified). Specificity ranged from 96.4%- 99.1% for SARS-CoV-2 antigen-specific IgA, with anti-Mt. Sinai IgA performing with the best specificity (99.1%; 105/106 negatives correctly classified).
  • sensitivity ranged from 2.2%-37.0% among serum samples collected ⁇ 10 days post symptom onset, and 28.9%-93.3% among serum samples collected >10 days post symptom onset (Fig. 5).
  • the best sensitivity was achieved by anti-Mt. Sinai RBD and anti- GenScript RBD (h) IgM, which both performed with 93.3% sensitivity (97/104 individuals with RT-qPCR confirmed prior SARS-CoV-2 infection correctly classified).
  • Specificity for serum IgM ranged from 96.2%-99.1%, with anti-Sino Biol. N and anti-GenScript RBD (i) IgM performing with 99.1% specificity (105/106 negatives correctly classified).
  • Temporal kinetics of SARS-CoV-2 antigen-specific IgG, IgA, and IgM responses in serum compared to saliva The temporal kinetics of antigen-specific IgG, IgA and IgM responses in serum and in saliva are shown in Fig. 6. Also shown are the cut-offs for each isotype (IgG, IgA and IgM) in serum and in saliva (dashed lines). The cut-offs were defined as the mean MFI signal plus three standard deviations using known negative serum and saliva samples. The temporal kinetics and magnitude of the antigen-specific IgG and IgA responses generally match those seen in serum.
  • the IgM response is significantly lower in saliva than in serum, which is consistent with the lower total IgM concentration in saliva compared to serum and compared to total IgA and IgG concentrations in saliva.
  • the IgA response across individuals consistently crosses the cut-off (dashed lines) several days before the IgG response. IgM and IgG seroconversion in serum seem to occur simultaneously.
  • IgA levels often do not cross the cut-off.
  • IgM levels in saliva are low and do not cross the cut-off of any antigen in the multiplex assay.
  • saliva the antigen-specific IgG response consistently crosses the cut-off approximately 10 days post symptom onset, i.e., roughly 15 days post infection, similar
  • Saliva anti-NAC SARS 2002 N IgG as a surrogate for SARS-CoV-2 N performed with sensitivity of 100% for detection of RT-qPCR confirmed prior SARS-CoV-2 infection among samples collected >10 days post symptom onset.
  • serum anti-NAC SARS 2002 N performed with a sensitivity of 88.5%
  • serum anti-NAC SARS 2002 N IgA with a sensitivity of 95.2%, for detection of RT-qPCR confirmed prior SARS-CoV-2 infection among samples collected >10 days post symptom onset.
  • the median MFI for saliva IgG and IgA, and serum IgG, IgA, and IgM, to NAC SARS 2002 N was significantly elevated among samples collected >10 days post symptom onset compared to negatives (Fig. 8 and Fig. 9).
  • the median MFI for saliva and serum IgG and IgA to Sino Biol. hCoV 229E ECD was also significantly elevated among samples collected >10 days post symptom onset compared to negatives (Fig. 8 and Figure 9).
  • Intra- and inter-assay variability Among 47 saliva samples run in duplicate on the same 96-well plate, the average intra-assay variability ranged from 2.66%-17.9% (CV%) (Table 3). Among 47 saliva samples run in duplicate on different 96-well plates on different days, the average inter-assay variability ranged from 4.60%-27.7% (CV%) (Table 3).
  • the data also show similar temporal kinetics of SARS-CoV-2 antigen-specific IgG responses in saliva as observed serum, where saliva and serum IgG responses peaked at ⁇ 10 days following RT-qPCR confirmed SARS-CoV-2 infection.
  • SARS-CoV-2 antigen-specific IgG responses in saliva reflect those observed in serum and can accurately identify subjects with prior SARS-CoV-2 infection.
  • This saliva-based multiplex immunoassay can serve as a non-invasive proxy to serum for accurate and large-scale SARS- CoV-2 “sero”-surveillance.
  • limited SARS-CoV-2 testing represents the “tip of the iceberg” with a large proportion of transmission undetected. The true population immunity and susceptibility to SARS-CoV-2 remains unclear.
  • results described herein are the first to measure SARS-CoV-2 antigen-specific IgG, IgA, and IgM responses in saliva, and compare these antibody responses to those observed in serum. While matched saliva and serum samples were limited in this study, the results show that SARS-CoV-2 antigen-specific IgG responses in saliva are strongly correlated with those in serum. The temporal kinetics of SARS-CoV-2 antigen-specific antibody responses in saliva following SARS-CoV-2 infection were also congruent with those observed in serum, and consistent with SARS-CoV-2 serum antibody responses reported in previous publications. First, synchronous elevation of SARS-CoV-2 antigen-specific IgG, IgA, and IgM responses were observed following SARS-CoV-2 infection.
  • RT-qPCR testing for SARS-CoV-2 was done ⁇ 4 days prior to symptom onset in most cases, a peak IgG response was observed at ⁇ 14 days post RT-qPCR confirmed SARS-CoV-2 infection, consistent with the previously reported median IgG and IgM seroconversion time of 13 days (Long Q-X, et al. Nat Med. 2020 Apr 29).
  • the sensitivity of the assay described herein improved, or stayed the same, among samples collected during convalescent phase (>10 days post symptom onset) compared to acute phase ( ⁇ 10 days post symptom onset) for the antigen-specific IgG, IgA, and IgM tested.
  • Saliva SARS-CoV-2 antigen-specific IgG peaked at 100% sensitivity, and serum SARS- CoV-2 antigen-specific IgG at 92.3% sensitivity (anti-Sino Biol. RBD and anti-Mt. Sinai RBD IgG, respectively) among samples collected >10 days post SARS-CoV-2 symptom onset.
  • Prior studies have reported sensitivities for various SARS-CoV-2 antibody tests peaking at 81.8%-100% sensitivity among samples collected during convalescent phase of infection (National COVID Testing Scientific Advisory Panel. Antibody testing for COVID- 19 : A report from the National COVID Scientific Advisory Panel. medRxiv. 2020; Lassauniere R, et al.
  • the lack of improved sensitivity for SARS-CoV-2 antigen specific IgA and IgM in saliva may be attributable to the saliva collection method used in this study, which specifically harvests gingival crevicular fluid enriched with primarily IgG antibody derived from serum (Brandtzaeg P. J Oral Microbiol. 2013;5(2013):l-24).
  • IgM antibodies are also not stable in saliva (BRANDTZAEG).
  • SARS-CoV-2 antigen-specific IgG responses in saliva performed with improved sensitivity and specificity than in serum, peaking at 100% sensitivity for anti-GenScript N IgG and 100% specificity for anti-Mt.
  • Sinai RBD IgG at >10 days post symptom onset.
  • Cross reactivity with antibodies from other coronavirus infections could lead to false positives.
  • the RBD of the S protein is less conserved across beta-CoVs than the N protein and whole S protein, and many antibodies known to interact with SARS-CoV-l’s RBD do not interact with SARS-CoV-2’s RBD (Tian X, et al. Emerg Microbes Infect. 2020 Jan l;9(l):382-5).
  • Cross-reactivity may largely be attributable to the N protein and S2 subunit, which share 90% sequence homology with SARS-CoV (Okba NMA, et al. Emerg Infect Dis. 2020 Jul;26(7)), supporting the use of SARS-CoV-Sl subunit or RBD for optimized specificity (Amanat F, et al. A serological assay to detect SARS-CoV -2 seroconversion in humans. medRxiv. 2020;2:2020.03.17.20037713). MERS-CoV S protein cross-reactive antibodies have also been detected in the serum of a COVID-19 confirmed patient, which was not seen when MERS-CoV SI was used for testing (Okba NMA, et al. Emerg Infect Dis.
  • SARS-CoV-2 antigen-specific antibody responses in saliva reflect those observed in serum, and that SARS-CoV-2 antigen-specific IgG can be used to accurately detect prior SARS-CoV-2 infection.
  • Described herein is a saliva-based multiplex immunoassay that was developed and validated to detect prior SARS-CoV-2 infection with 100% sensitivity (anti-N IgG) and 100% specificity (anti-RBD IgG) at >10 days post symptom onset.
  • the methods disclosed herein can be used to improve individual and population health for those at risk for SARS-CoV-2 infection.
  • Example 2 Algorithms to identify individuals with prior COVID-19 infection based on salivary anti-SARS-CoV-2 IgG levels
  • the IgG signal must be higher than that observed among negative (e.g., pre-COVID-19 era) samples. Cutoffs (median fluorescence intensity or MFI values) for each antigen are defined as the mean IgG signal (observed MFI) against each antigen of negative saliva samples tested (pre-COVID-19 era; 134 negative saliva samples) plus three standard deviations.
  • Negative saliva samples must be qualified as containing total antibody levels in the normal range by testing total IgG, IgA or IgM to assure the negative signal is not due to a degraded sample or due to a too diluted sample, e.g., because the participant just drank a lot of water.
  • the cutoffs are shown in Table 5.
  • Cutoffs shown are calculated after subtracting IgG MFI to BSA to account for nonspecific binding to the included BSA control bead set for each saliva sample.
  • the sample set included 134 negative saliva samples and 33 positive saliva samples (see, Table 6). Positive saliva samples were collected from subjects that were clinically confirmed to have been infected with SARS-CoV-2 by RT-PCR. The clinical status (SARS- CoV-2 positive by RT-PCR) serves as reference to calculate sensitivity. Pre-COVID-19 samples (negative samples) and samples from patients who were clinically determined to not have been infected with SARS-CoV-2 by RT-PCR and/or blood antibody test and/or clinical presentation were used to calculate specificity of the method (e.g., salivary IgG test). Among the positive samples, 28 were collected 10 or more days after COVID-19 symptom onset. Saliva samples included in these analyses are shown in Table 6; additional “days between saliva collection and symptom onset” information is shown in Table 7.
  • Table 6 Samples collected and included.
  • Table 7. SARS-CoV-2 RT-PCR distribution by days since symptom onset.
  • saliva samples from SARS-CoV-2 RT-PCR positive individuals were collected 10+ days post symptom onset. From the data, algorithms (non-exhaustive) were identified that can classify the saliva samples that were collected at least 10 days after reported COVID-19 symptom onset correctly as positive and thus the samples that do not meet these criteria as negative. These algorithms as shown in Table 8 resulted in 100% sensitivity for samples collected 10+ days post symptom onset and 100% specificity for pre-COVID-19 era and clinically confirmed negative saliva samples.
  • Tables 9-12 show the results of the algorithms tests tested.
  • Table 9 shows the result for Algorithm 1: Positive for GenScript N and for Sino Biol. RBD or Sino Biol. ECD.
  • Table 10 shows the result for Algorithm 2: Positive for GenScript N and for Mt. Sinai RBD or Sino Biol. ECD.
  • Table 11 shows the result for Algorithm 3: Positive for GenScript N and for GenScript RBD (h) or Sino Biol. ECD.
  • Table 12 shows the result for Algorithm 4: Positive for GenScript N and for
  • Negative samples One replicate each of 10 saliva samples collected prior to the COVID-19 pandemic were tested. The result (combined index) ranged between 0.3 and 1.6, well below the cutoff of 10. The samples were correctly classified as negative.
  • the SARS-CoV-2 IgG saliva test is an indirect immunoassay based on Luminex xMAP technology.
  • the test measures binding of salivary IgG to multiple SARS-CoV-2 antigens (two nucleocapsid [N], three receptor binding domain [RBD] antigens, one Spike [S] 1 / Spike 2 / ectodomain and one whole Spike antigen) in a single test in a 96-well microplate format.
  • Saliva collected with an Oracol S14 saliva collection device is added to assay buffer containing SARS-CoV-2 antigens, which have been covalently coupled to color-coded magnetic microparticles (Luminex MagPlex microspheres or “beads”).
  • the saliva sample is incubated with the magnetic beads.
  • IgG to SARS-CoV-2 present in the sample will bind to the SARS-CoV-2 antigens on magnetic beads. Beads are then separated magnetically and washed twice to remove any components that did not bind to the beads.
  • a detectable antibody (phycoerythrin [PE] -labeled anti -human IgG antibody) can be added.
  • Luminex MAGPIX instrument with xPONENT software.
  • This instrumentation uses LED excitation and a CCD camera to read out emission of fluorescence signals.
  • a standard curve ranging from ⁇ 5 to 10,000 arbitrary units (au) is included on each plate.
  • the signal (au) to cutoff for IgG binding to each antigen is calculated and samples are classified as “positive” if the combined signal to cutoff value ( ⁇ (S/Co)) of the antigens crosses the threshold or “negative” if the combined signal to cutoff is below that threshold. If samples test negative, an ELISA that measures total salivary IgG must be performed to confirm whether the sample had enough total IgG (minimum acceptable concentration is 15 pg/mL).
  • the sum of the signal to cutoffs ⁇ (S/Co) of the seven SARS-CoV-2 beads for each sample was calculated and compared to the ⁇ (S/Co) of presumed positive samples.
  • An overall combined ⁇ (S/Co) of 10 resulted in 100% specificity (219/219) and 97% sensitivity (118/222) using samples collected >14 days post COVID-19 symptoms onset (DPSO) after excluding samples with ⁇ (S/Co)

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L'invention concerne des procédés de détection d'un ou de plusieurs anticorps anti-coronavirus seule ou en combinaison avec la détection d'anticorps anti-virus respiratoires dans un échantillon de fluide buccal avec une spécificité et une sensibilité élevées.
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